Deletion of tetraspanin Cd151 results in decreased pathologic angiogenesis in vivo and in vitro

Tetraspanin protein CD151 is abundant on endothelial cells. To determine whether CD151 affects angiogenesis, Cd151-null mice were prepared. Cd151-null mice showed no vascular defects during normal development or during neonatal oxygen-induced retinopathy. However, Cd151-null mice showed impaired pathologic angiogenesis in other in vivo assays (Matrigel plug, corneal micropocket, tumor implantation) and in the ex vivo aortic ring assay. Cd151-null mouse lung endothelial cells (MLECs) showed normal adhesion and proliferation, but marked alterations in vitro, in assays relevant to angiogenesis (migration, spreading, invasion, Matrigel contraction, tube and cable formation, spheroid sprouting). Consistent with these functional impairments, and with the close, preferential association of CD151 with laminin-binding integrins, Cd151-null MLECs also showed selective signaling defects, particularly on laminin substrate. Adhesion-dependent activation of PKB/c-Akt, e-NOS, Rac, and Cdc42 was diminished, but Raf, ERK, p38 MAP kinase, FAK, and Src were unaltered. In Cd151-null MLECs, connections were disrupted between laminin-binding integrins and at least 5 other proteins. In conclusion, CD151 modulates molecular organization of laminin-binding integrins, thereby supporting secondary (ie, after cell adhesion) functions of endothelial cells, which are needed for some types of pathologic angiogenesis in vivo. Selective effects of CD151 on pathologic angiogenesis make it a potentially useful target for anticancer therapy.     

Elevated matrix metalloprotease and angiostatin levels in integrin alpha 1 knockout mice cause reduced tumor vascularization

Integrin alpha1beta1 is a collagen receptor abundantly expressed on microvascular endothelial cells. As well as being the only collagen receptor able to activate the Ras/Shc/mitogen-activated protein kinase pathway promoting fibroblast cell proliferation, it also acts to inhibit collagen and metalloproteinase (MMP) synthesis. We have observed that in integrin alpha1-null mice synthesis of MMP7 and MMP9 was markedly increased compared with that of their wild-type counterparts. As MMP7 and MMP9 have been shown to generate angiostatin from circulating plasminogen, and angiostatin acts as a potent inhibitor of endothelial cell proliferation, we determined whether tumor vascularization was altered in the alpha1-null mice. Tumors implanted into alpha1-null mice showed markedly decreased vascularization, with a reduction in capillary number and size, which was accompanied by an increase in plasma levels of angiostatin due to the action of MMP7 and MMP9 on circulating plasminogen. In vitro analysis of alpha1-null endothelial cells revealed a marked reduction of their proliferation on both integrin alpha1-dependent (collagenous) and independent (noncollagenous) substrata. This reduction was prevented by culturing alpha1-null cells with plasma derived from plasminogen-null animals, thus omitting the source from which to generate angiostatin. Plasma from tumor-bearing alpha1-null animals uniquely inhibited endothelial cell growth, and this inhibition was relieved by the coaddition of either MMP inhibitors, or antibody to angiostatin. Integrin alpha1-deficient mice thus provide a genetically characterized model for enhanced angiostatin production and serve to reveal an unwanted potential side effect of MMP inhibition, increased tumor angiogenesis.     

alpha2beta1 integrin expression in the tumor microenvironment enhances tumor angiogenesis in a tumor cell-specific manner

To define the role of the alpha2beta1 integrin in pathologic angiogenesis, we investigated tumor-associated growth and angiogenesis in wild-type and alpha2-null mice. Our findings reveal that the alpha2beta1 integrin plays an important role in angiogenesis via regulation of VEGFR1 expression. When challenged with B16F10 melanoma cells, mice lacking alpha2beta1 integrin ex-pression exhibit increased tumor angiogenesis associated with up-regulated VEGFR1 expression. In contrast, there was no alpha2beta1 integrin-dependent difference in the angiogenic response to Lewis lung carcinoma (LLC) cells. Interestingly, whereas B16F10 cells secrete high levels of placental growth factor (PLGF), LLC cells produce high levels of VEGF, but low levels of PLGF. The alpha2beta1 integrin-dependent difference in angiogenesis was restored to LLC cells by expression of PLGF, strongly suggesting that the angiogenic phenotype and tumor growth in the alpha2-null host is dependent on specific interactions between the tumor cell and the genetically defined integrin repertoire of the host microenvironment. Thus integrin alpha2-null mice represent an example of genetic alterations of "the soil" determining response to the "seed."     

Increased primary tumor growth in mice null for beta3- or beta3/beta5-integrins or selectins

Expression of alphavbeta3- or alphavbeta5-integrins and selectins is widespread on blood cells and endothelial cells. Here we report that human tumor cells injected s.c. into mice lacking beta3- or beta3/beta5-integrins or various selectins show enhanced tumor growth compared with growth in control mice. There was increased angiogenesis in mice lacking beta3-integrins, but no difference in structure of the vessels was observed by histology or by staining for NG2 and smooth muscle actin in pericytes. Bone marrow transplants suggest that the absence of beta3-integrins on bone marrow-derived host cells contributes to the enhanced tumor growth in beta3-null mice, although few, if any, bone marrow-derived endothelial cells were found in the tumor vasculature. Tumor growth also was affected by bone marrow-derived cells in mice lacking any one or all three selectins, implicating both leukocyte and endothelial selectins in tumor suppression. Reduced infiltration of macrophages was observed in tumors grown in mice lacking either beta3-integrins or selectins. These results implicate cells of the innate immune system, macrophages or perhaps natural killer cells, in each case dependent on integrins and selectins, in tumor suppression.     

Impaired tumor growth,metastasis, angiogenesis and wound healing in annexin A1-null mice

Despite 2 decades of research, no clear function for annexin A1 (AnxA1) has been established. Using AnxA1-KO mice, we show that tumor growth and metastasis are significantly decreased, whereas rodent survival and tumor necrosis are greatly increased when tumors grow in AnxA1-KO mice. Systems analysis of gene expression in these tumors specifically implicates 2 related vascular functions, angiogenesis and wound healing, in this impairment. Both tumor vascular development and wound healing are greatly retarded in KO tissues. Aortic ring assays reveal induced AnxA1 expression on sprouting endothelial cells of normal mice whereas KO aortas exhibit impaired endothelial cell sprouting that is rescued by adenoviral expression of AnxA1. Key differences in specific gene regulation may define new molecular pathways mediating angiogenesis, including a reset profile of pro- versus anti-angiogenic factors, apparently distinct for physiological versus pathological angiogenesis. These studies establish novel pro-angiogenic functions for AnxA1 in vascular endothelial cell sprouting, wound healing, and tumor growth and metastasis, thereby uncovering a new functional target for repairing damaged tissue and treating diseases such as cancer. They also provide critical new evidence that the tumor stroma and its microenvironment can greatly affect tumor progression and metastasis.     

Diminished metastasis in tetraspanin CD151-knockout mice

Tetraspanin protein CD151 on tumor cells supports invasion and metastasis. In the present study, we show that host animal CD151 also plays a critical role. CD151-null mice showed markedly diminished experimental lung metastasis after injection of Lewis lung carcinoma or B16F10 melanoma cells. Diminished tumor cell residence in the lungs was evident 6-24 hours after injection. Consistent with an endothelial cell deficiency, isolated CD151-null mouse lung endothelial cells showed diminished support for B16F10 adhesion and transendothelial migration, diminished B16F10-induced permeability, and diminished B16F10 adhesion to extracellular matrix deposited by CD151-null mouse lung endothelial cells. However, CD151 deletion did not affect the size of metastatic foci or subcutaneous primary B16F10 tumors, tumor aggregation, tumor clearance from the blood, or tumor-induced immune cell activation and recruitment. Therefore, the effects of host CD151 on metastasis do not involve altered local tumor growth or immune surveillance. VEGF-induced endothelial cell signaling through Src and Akt was diminished in CD151-null endothelial cells. However, deficient signaling was not accompanied by reduced endothelial permeability either in vitro (monolayer permeability assay) or in vivo (VEGF-stimulated Miles assay). In summary, diminished metastasis in CD151-null host animals may be due to impaired tumor-endothelial interactions, with underlying defects in mouse lung endothelial cell extracellular matrix production.     

Impaired angiogenesis in glutathione peroxidase-1-deficient mice is associated with endothelial progenitor cell dysfunction

Several vascular disease are characterized by elevated levels of reactive oxygen species (ROS). Vascular endothelium is protected from oxidant stress by expressing enzymes such as glutathione peroxidase type 1 (GPx-1). In this study, we investigated the effect of vascular oxidant stress on ischemia-induced neovascularization in a murine model of homozygous deficiency of GPx-1. GPx-1-deficient mice showed impaired revascularization following hindlimb ischemic surgery based on laser Doppler measurements of blood flow and capillary density in adductor muscle. GPx-1-deficient mice also showed an impaired ability to increase endothelial progenitor cell (EPC) levels in response to ischemic injury or subcutaneous administration of vascular endothelial growth factor protein. EPCs isolated from GPx-1-deficient mice showed a reduced ability to neutralize oxidative stress in vitro, which was associated with impaired migration toward vascular endothelial growth factor and increased sensitivity to ROS-induced apoptosis. EPCs isolated from GPx-1-deficient mice were impaired in their ability to promote angiogenesis in wild-type mice, whereas wild-type EPCs were effective in stimulating angiogenesis in GPx-1-deficient mice. These data suggest that EPC dysfunction is a mechanism by which elevated levels of ROS can contribute to vascular disease.     

Function of endogenous inhibitors of angiogenesis as endothelium-specific tumor suppressors

Disruption of the systemic angiogenesis balance to favor enhanced angiogenesis is speculated to represent a key step in the growth of tumors. Although a major emphasis has been placed on the increase of angiogenesis stimulators, such as VEGF, on the disruption of the angiogenic balance, the potential role of the physiological levels of endogenous inhibitors of angiogenesis on tumor growth is poorly understood. Here, we use three independent lines of mice deficient in tumstatin, endostatin, or thrombospondin-1 (TSP-1), to address the role that these endogenous angiogenesis inhibitors play in tumor growth. Our experiments demonstrate that normal physiological levels of these inhibitors serve to retard the growth of tumors, and that their absence leads to enhanced angiogenesis and a 2- to 3-fold increase in tumor growth. The tumor-suppressive action of TSP-1, endostatin, and tumstatin correlates with expression of CD36 receptor, alpha5beta1 integrin, and alphavbeta3 integrin on proliferating endothelial cells, respectively. Moreover, tumors grow 2-fold faster in the tumstatin/TSP-1 double-knockout mice, compared with either the tumstatin- or the TSP-1-deficient mice, strongly suggesting that ceiling rate of cancer growth is not completely dependent on the genetic defects of cancer cells but also depends on the host-derived tumor microenvironment. Additionally, tumor growth in transgenic mice overproducing endostatin specifically in the endothelial cells (a 1.6-fold increase in the circulating levels; mimicking Down's syndrome condition) is 3-fold slower than the tumor growth in wild-type mice. Collectively, our data suggest that physiological levels of endogenous inhibitors of angiogenesis can serve as endothelium-specific tumor suppressors.     

Nucleolin is a receptor that mediates antiangiogenic and antitumor activity of endostatin

The exact molecular mechanism of how endostatin inhibits angiogenesis and tumor growth remains uncharacterized. Here, we report that endostatin specifically binds to the cell surface nucleolin with high affinity. Blockage of nucleolin by a neutralizing antibody or knockdown of nucleolin by the RNA interference results in loss of antiendothelial activities of endostatin. Importantly, a neutralizing antinucleolin antibody abrogates the antiangiogenic and antitumor activities of endostatin in vivo. Nucleolin and endostatin are colocalized on the cell surface of endothelial cells of angiogenic blood vessels in the tumor environment. Finally, we found that endostatin is internalized and transported into cell nuclei of endothelial cell via nucleolin. In the nucleus, the phosphorylation of nucleolin, which is critical for cell proliferation, can be inhibited by endostatin. Our studies demonstrate that nucleolin is a novel functional receptor for endostatin, and mediates the antiangiogenic and antitumor activities of endostatin. These findings also provide mechanistic insights of how endostatin specifically inhibits proliferating endothelial cell growth and angiogenesis.     

Examining the role of Rac1 in tumor angiogenesis and growth: a clinically relevant RNAi-mediated approach

Angiogenesis, the sprouting of new blood vessels from the pre-existing vasculature, is a well established target in anti-cancer therapy. It is thought that the Rho GTPase Rac1 is required during vascular endothelial growth factor (VEGF)-mediated angiogenesis. In the present study, we have used a clinically relevant RNA interference approach to silence Rac1 expression. Human umbilical vein endothelial cells were transiently transfected with non-specific control siRNA (siNS) or Rac1 siRNA (siRac1) using electroporation or Lipofectamine 2000. Functional assays with transfected endothelial cells were performed to determine the effect of Rac1 knockdown on angiogenesis in vitro. Silencing of Rac1 inhibited VEGF-mediated tube formation, cell migration, invasion and proliferation. In addition, treatment with Rac1 siRNA inhibited angiogenesis in an in vivo Matrigel plug assay. Intratumoral injections of siRac1 almost completely inhibited the growth of grafted Neuro2a tumors and reduced tumor angiogenesis. Together, these data indicate that Rac1 is an important regulator of VEGF-mediated angiogenesis. Knockdown of Rac1 may represent an attractive approach to inhibit tumor angiogenesis and growth.     

Antibody against murine PECAM-1 inhibits tumor angiogenesis in mice

Platelet endothelial cell adhesion molecule (PECAM-1/CD31), a member of the immunoglobulin superfamily expressed at high levels on endothelial cells, has been recently implicated in angiogenesis. Although antagonism of PECAM-1 inhibited neovascularization in two different animal models of growth factor/chemokine-induced angiogenesis, its participation in tumor angiogenesis has not been established. We therefore investigated its involvement in models of tumor angiogenesis in mice. An antibody against murine PECAM-1 that was shown to block in vitro murine endothelial tube formation inhibited the subcutaneous growth and tumor vascularity of three tumors in mice: A549 human non-small cell lung cancer in SCID mice, B16 murine melanoma in C57BL/6 mice and AB12 murine mesothelioma in Balb/c mice. These studies suggest a possible role for PECAM-1 in the complex process of tumor angiogenesis and provide additional evidence of the importance of endothelial cell adhesion molecules to the formation of new vessels. This revised version was published online in June 2006 with corrections to the Cover Date.     

CLT1 targets angiogenic endothelium through CLIC1 and fibronectin

Angiogenesis is important for tumor growth and metastasis. CLT1 (CGLIIQKNEC), a peptide that binds to tumor interstitial spaces in the presence of fibrin-fibronectin, has structural similarity to the anti-angiogenic β-sheet peptides anastellin and anginex. This similarity is reflected in the ability of CLT1 to form co-aggregates with fibronectin that induce an unfolded protein response and cause autophagic cell death in proliferating endothelial cells. CLT1 cytotoxicity is mediated at least in parts by a novel CLT1 binding protein, Chloride Intracellular Channel 1 (CLIC1), which promotes internalization of CLT1-fibronectin co-aggregates in a mechanism that depends on the LIIQK amino acid sequence of CLT1. LIIQK encompasses amino acid residues relevant for CLT1 binding to CLIC1 and in addition, facilitates the formation of CLT1-fibronectin co-aggregates, which in turn promote translocation of CLIC1 to the endothelial cell surface through ligation of integrin αvβ3. Paralleling the in vitro results, we found that CLT1 co-localizes with CLIC1 and fibronectin in angiogenic blood vessels in vivo, and that CLT1 treatment inhibited angiogenesis and tumor growth. Our findings show that CLT1 is a new anti-angiogenic compound, and its mechanism of action is to form co-aggregates with fibronectin, which bind to angiogenic endothelial cells through integrins, become internalized through CLIC1 and elicit a cytotoxic unfolded protein response. The simple structure and high potency of CLT1 make it a potentially useful compound for anti-angiogenic treatments.     

Reduced angiogenic responses in adult Endoglin heterozygous mice

OBJECTIVE: To determine if angiogenesis is altered in adult Endoglin heterozygous (Eng(+/-)) mice, the animal model for the vascular disorder hereditary hemorrhagic telangiectasia type 1 (HHT1). METHODS: Primary cultures of endothelial cells were generated from Eng(+/-) and Eng(+/+) mice and analyzed for proliferation, migration, and ability to form capillary-like tubes. Endothelial cells derived from umbilical veins of newborns (HUVEC) with an HHT1 genotype were also tested for capillary formation. Two in vivo models of angiogenesis were tested in the Eng(+/-) and Eng(+/+) mice: Matrigel implant-dependent angiogenesis and reperfusion following hindlimb ischemia. RESULTS: The Eng(+/-) endothelial cells displayed significantly reduced proliferation and migration, increased collagen production, and decreased NO synthase expression and vascular endothelial growth factor (VEGF) secretion. They also showed impaired capillary tube formation in vitro, as did the HHT1 HUVEC. These endothelial cell-specific abnormalities were associated with impaired Matrigel-dependent capillary tube formation in vivo and delayed reperfusion following hindlimb ischemia. CONCLUSIONS: Although vascular development is normal in Eng(+/-) mice, angiogenic abnormalities were observed in the adult mice and their isolated endothelial cells. These results suggest that a normal level of endoglin is required for full angiogenic activity.     

Endoglin in angiogenesis and vascular diseases

Endoglin is a transmembrane auxillary receptor for transforming growth factor-β (TGF-β) that is predominantly expressed on proliferating endothelial cells. Endoglin deficient mice die during midgestation due to cardiovascular defects. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-β type I receptor, have been linked to hereditary hemorrhagic telangiectasia (HHT), an autosomal dominant vascular dysplasia characterized by telangiectases and arteriovenous malformations. Endoglin heterozygote mice develop HHT-like vascular abnormalities, have impaired tumor and post-ischemic angiogenesis and demonstrate an endothelial nitric oxide synthase-dependent deterioration in the regulation of vascular tone. In pre-eclampsia, placenta-derived endoglin has been shown to be strongly upregulated and high levels of soluble endoglin are released into the circulation. Soluble endoglin was found to cooperate with a soluble form of vascular endothelial growth factor receptor 1 in the pathogenesis of pre-eclampsia by inducing endothelial cell dysfunction. Endoglin is highly expressed in tumor-associated endothelium, and endoglin antibodies have been successfully used to target activated endothelial cells and elicit anti-angiogenic effects in tumor mouse models. These exciting advances provide opportunities for the development of new therapies for diseases with vascular abnormalities.     

Antiangiogenesis therapy for endometriosis

It is known that angiogenesis is of pivotal importance for the development of endometriosis. However, in the treatment of endometriosis patients, prevention of endometriosis lesion development only will not be sufficient as a therapy. Treatment options, aimed at interfering with established lesions, have to be developed. In this study we evaluated whether inhibition of angiogenesis by angiostatic therapy is also effective in antagonizing the sustentation of endometriosis. We evaluated the effect of the angiostatic compounds antihuman vascular endothelial growth factor, TNP-470, endostatin, and anginex on the growth of established endometriosis lesions in the nude mouse model. We show that human endometrium in the proliferative endometrium is highly angiogenic and that vascular endothelial growth factor-A is the most important angiogenesis promotory factor. The angiostatic compounds significantly decreased microvessel densities and the number of established endometriosis lesions. In the remaining lesions, the number of pericyte-protected vessels is not different in control and treated mice; however, the number of unprotected vessels was significantly reduced in the groups treated with the angiostatic agents. Our data demonstrate that inhibitors of angiogenesis effectively interfere with the maintenance and growth of endometriosis by inhibiting angiogenesis. This suggests that the use of angiostatic agents may be promising as a therapy for endometriosis.     

Kallistatin is a new inhibitor of angiogenesis and tumor growth

Kallistatin is a unique serine proteinase inhibitor (serpin) and a heparin-binding protein. It has been localized in vascular smooth muscle cells and endothelial cells of human blood vessels, suggesting that kallistatin may be involved in the regulation of vascular function. Our previous study showed that kallistatin plays a role in neointima hyperplasia. In this study, we investigated the potential role of kallistatin in angiogenesis in vitro and in vivo. Purified human kallistatin significantly inhibited vascular endothelial growth factor (VEGF)- or basic fibroblast growth factor (bFGF)-induced proliferation, migration, and adhesion of cultured endothelial cells. Kallistatin attenuated VEGF- or bFGF-induced capillary density and hemoglobin content in subcutaneously implanted Matrigel plugs in mice. To further investigate the role of kallistatin in angiogenesis, we prepared adenovirus carrying the human kallistatin cDNA (Ad.HKBP) and evaluated the effect of kallistatin gene delivery on spontaneous angiogenesis in a rat model of hind-limb ischemia. Local kallistatin gene delivery significantly reduced capillary formation and regional blood perfusion recovery in the ischemic hind limb after removal of the femoral artery. Furthermore, a single intratumoral injection of Ad.HKBP into pre-established human breast tumor xenografts grown in athymic mice resulted in significant inhibition of tumor growth. CD31 immunostaining of tumor sections showed a decreased number of blood vessels in the kallistatin-treated group as compared to the control. These results demonstrate a novel role of kallistatin in the inhibition of angiogenesis and tumor growth.     

A novel role for inducible Fut2 in angiogenesis

Rationale

Angiogenesis plays an important role in wound healing and tumor growth. Fucosyltransferases synthesize fucosylated glycans and may play a major role in vascular biology.

Objective

To examine the role of an alpha(1,2) fucosyltransferase (Fut2) in angiogenesis.

Methods and results

We found that Fut2 mRNA and protein expression is inducible in human dermal microvascular endothelial cells (HMVECs). After finding that Fut2 is inducible in HMVECs, we examined if Fut2 contributes to angiogenesis. We found that Fut2 null endothelial cell (EC) migration and tube formation were significantly less compared to wild type (wt) ECs. Angiogenesis was impaired in Fut2 null compared to wt mice in the mouse Matrigel plug and the sponge granuloma angiogenesis assays. To assess the characteristics of Fut2 null ECs in vivo, we performed Matrigel plug angiogenesis assays in wt mice using Fut2 null and wt mouse ECs. We found a significant decrease in Fut2 null EC incorporation in neoangiogenesis compared to wt ECs. ERK1/2 activation, fibroblast growth factor receptor2, and vascular endothelial growth factor expression were less in Fut2 null ECs, suggesting a possible mechanism of impaired angiogenesis when Fut2 is lacking.

Conclusions

These data suggest a novel role for Fut2 as a regulator of angiogenesis.     

Structure and function of placental growth factor

Placental growth factor (PlGF) belongs to the same family as the vascular endothelial growth factor A (VEGF-A). Recent gene inactivation studies in mice have demonstrated that loss of PlGF does not affect development, reproduction, or normal postnatal life. However, the mice show significantly impaired angiogenesis and arteriogenesis during pathological conditions such as ischemia and tumor formation, conditions in which the expression of VEGF-A is normally increased. Mice expressing a truncated form of the specific receptor for PlGF, the VEGF receptor 1 (VEGFR-1), show impaired angiogenesis similar to that observed in Plgf(-/-)mice. These data suggest a pivotal role for PlGF and VEGFR-1 in regulating VEGF-A-dependent angiogenesis under pathological conditions. VEGF-A has been utilized for the therapeutic stimulation of new blood vessels in ischemic hearts and limbs, with controversial results from the initial clinical experience. This review discusses the possibility of using the PlGF/VEGFR-1 pathway as an alternative target for angiogenic therapy.